Toll-like receptors (TLRs) are type-I transmembrane proteins which are responsible for initiation of innate immune responses in vertebrates. They recognize a variety of pathogen-associated molecular patterns (PAMPS) from bacteria, viruses and fungi and act as a first line of defense against invading pathogens. There are ten human TLRs that elicit overlapping yet distinct biological responses due to differences in cellular expression and signaling pathways they initiate.
TLRs possess 19-25 contiguous copies of an extracellular N-terminal motif known as the leucine-rich repeat (LRR) motif, followed by a cysteine-rich region and an intracellular region. (Bell et al., Trends Immunol. 24:528 (2003)). The LRR region is a common feature among the TLRs and is important for ligand binding and cellular signaling. (Modlin, R. L., Ann. Allergy Asthma Immunol. 88:543 (2002); Kobe and Diesenhofer, Curr. Opin. Struct. Biol. 5:409 (1995)).
In humans, TLR7 is expressed mostly in human plasmocytoid dendritic cells (pDCs) and B cells. TLR8 is expressed mostly in cells of myeloid origin, i.e., monocytes, granulocytes and myeloid dendritic cells. TLR9, like TLR7, is expressed in B cells and dendritic cells. (Zarember and Godowski, J. Immunol. 168:554 (2002); Hornung et al., J. Immunol. 168:4 (2002); Rehli, Trends Immunol. 23:375 (2002).
TLR9, which appears to be localized intracellularly, is involved in the inflammatory response to bacterial DNA and oligonucleotides that contain unmethylated CpG sequences. (Shirota, H. et al., J. Immunol. 167:1882 (2001); Takeda, K. et al., Annu. Rev. Immunol. 21:335 (2003)). The role of TLR7, and of its close homologue TLR8, is to detect the presence of “foreign” single-stranded RNA within a cell, as a means to respond to viral invasion. (Heil et al., Science 303:1526 (2004); Diebold et al., Science 303:1529 (2004)). Single-stranded RNA derived from HIV-1 or the influenza virus was shown to induce the production of proinflammatory cytokines in pDCs. The distinguishing features of viral RNAs might be either their high U or G/U content, or the absence of a long poly-A tail at the 3′ terminus of messenger RNA.
Certain compounds of the imidazoquinoline family, notably R848, act as agonists for TLR7 and TLR8. (Heine and Lein, Int. Arch. Allergy Immunol. 130:180 (2003); Dunne and O'Neill, Sci. STKE 2003:re3 (2003)). Once engaged, TLRs initiate a signal transduction cascade leading to activation of NFκB via the adapter protein myeloid differentiation primary response gene 88 (MyD88) and recruitment of the IL-1 receptor associated kinase (IRAK). Phosphorylation of IRAK then leads to recruitment of TNF-receptor associated factor 6 (TRAF6), which then results in the phosphorylation of the NFκB inhibitor I-κB. As a result, NFκB enters the cell nucleus and initiates transcription of genes whose promoters contain NFκB binding sites, such as cytokines (Singh et al., Curr. Sci. 85:1156 (2003)). Indeed, treatment of TLR8-expressing cells, such as PBMCs, with R848 results in production of high levels of IL-12, IFN-γ, IL-1, TNF-α, IL-6 and other inflammatory cytokines. Similarly, stimulation of TLR7-expressing cells, such as pDCs, results in production of very high levels of interferon-α and low levels of inflammatory cytokines.
Through activation of dendritic cells and other antigen-presenting cells, TLR7, TLR8 or TLR9 engagement and cytokine production is expected to activate diverse innate and acquired immune response mechanisms leading to the destruction of pathogens, infected cells or tumor cells. Thus, there is a need for agonists of TLR7, TLR8 or TLR9 for the treatment of cancer, virus infections, allergy, asthma, and COPD. TLR7, TLR8 or TLR9 and agonists thereof, however, require relatively high concentrations of compounds (in the double digit μg/ml range), and such concentrations are often difficult to achieve with complex chemical compounds due to solubility and/or cytotoxicity issues. As a result, screening is usually performed under conditions of low compound concentrations where possible agonists might not give activation levels readily detectable using standard cellular assays for screening. Thus, there is an immediate need for improved screening methods for identifying TLR7, TLR8 or TLR9 agonists.